In the past several years we utilized l qualitative and quantitative proteomic technology to analyze protein expression in whole cell and subcellular fractions of several pathogenic bacteria such as Pseudomonas aeruginosa, Salmonella typhimurium and Francisella novicida. The goal of our studies was to identify bacterial virulence components by monitoring differential protein expression in bacteria grown under conditions that induce expression of virulence factors. Quantitative proteome analysis was performed after differential stable isotope labeling of whole cell proteins by alkylation of cysteinyl residues with isotope-coded affinity tags (ICAT). This method offers several advantages over proteome analysis by two-dimensional gel electrophoresis. ICAT-labeled peptides are analyzed using microcapillary liquid chromatography-electrospray ionization-tandem mass spectrometry (?LC-ESI-MS/MS), and differential protein abundance is determined by using protein identification and quantification software.
Goal of one study is to identify P. aeruginosa proteins that are commonly expressed in isolates from lungs of cystic fibrosis (CF) patients. Recently, our laboratory has fully analyzed the proteome of P. aeruginosa and quantified protein changes after bacterial growth in media of different magnesium concentrations that mimic selected characteristics of the CF airway. All previously defined P. aeruginosa magnesium-stress response proteins were identified, confirming the validity of this approach. Furthermore, this study demonstrated that even small (such as 1.5- to 2-fold) changes in protein abundance could have significant biological effects as observed for the enzymes of the biosynthetic pathways for the P. aeruginosa quorum sensing signaling quinolone molecules (PQS). Quorum sensing is a mode of bacterial intercellular communication that regulates P. aeruginosa virulence factors as well as production of biofilms, antibiotic-resistant bacterial communities. This data allowed us to define increased production of PQS by P. aeruginosa clinical isolates from infants with CF. Analysis of PQS production in about 200 P. aeruginosa clinical isolates established that the increased production of PQS might be one of the early mechanisms for adaptation of P. aeruginosa to the CF lung.
Significant Gram-negative envelope remodeling is part of bacterial adaptation to magnesium limitation. To define proteins that contribute to the envelope remodeling either structurally or by providing an enzymatic function to this process by mediating LPS modifications, total membrane protein of P. aeruginosa was also analyzed by quantitative proteomic analysis. Results of our proteomic analysis using differential labeling with ICAT and comparison of the protein relative abundance ratios to those obtained for the whole cell protein in the same growth condition revealed that twenty P. aeruginosa proteins were shifting subcellular compartment during bacterial growth in magnesium-limited medium.
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